Peptide ligands for affinity purification of human Factor VIII

ABSTRACT

Peptides which bind Factor VIII are disclosed. The peptides have available Factor VIII binding domains having a Trp-His-Tyr-Tyr-His-Gly, His-Ile-Gln-His-Tyr-His, or His-Gln-Tyr-Gly-Tyr-His sequence. Peptides having at least one of these Factor VIII binding domains are immobilized upon a chromatographic substrate in a preferred embodiment of the invention. This preferred embodiment is useful in a chromatography process to purify human Factor VIII.

BACKGROUND OF THE INVENTION

1. Field

This invention is generally directed to identifying protein-ligandinteractions, and specifically with peptide ligands which bind humanFactor VIII and which may be used in a process for the affinitypurification of human Factor VIII.

2. Background

Human Factor VIII, (antihemophilic factor; FVIII:C) is a human plasmaprotein consisting of 2 polypeptides (light chain molecular weight of80,000 daltons and heavy chain molecular weight variable from 90,000 to220,000). It is an essential cofactor in the coagulation pathway;required for the conversion of Factor X into its active form (FactorXa). Factor VIII circulates in plasma as a non-covalent complex with vonWillibrand Factor (aka FVIII:RP). Blood concentrations of Factor VIIIbelow 20% of normal cause a bleeding disorder designated hemophilia A.Factor VIII blood levels less than 1% result in a severe bleedingdisorder, with spontaneous joint bleeding being the most common symptom.Factor VIII can be isolated from either a plasma derived source(cryoprecipitate) or from a genetically engineered recombinant source.Recombinant DNA technology has allowed construction of plasmids thatdirect the expression of fusion products of Factor VIII protein intransfected mammalian cells (See U.S. Pat. No. 4,757,006).

Several methods have been described for purification of Factor VIII fromplasma sources. Tuddenham et al. disclose a method for separating FVIIIfrom human plasma by immunoabsorbent chromatography (Tuddenham, et al.,J. Laboratory Clin. Med., (1979), 93: 40-53). The method involved usingrabbit anti-FVIII:C polyclonal antibodies adsorbed to agarose beads, anddesorption using a calcium gradient. Notably, it was sufficientlyselective to distinguish FVIII:C from FVIII:RP. D.E.G. Austendemonstrated another technique using ion-exchange chromatography onamino-hexyl-substituted agarose beads (Austin, D. E. G., British J.Heamotol., (1979), 43: 669-674). However, it has been reported that bothmethods suffer from some level of contamination of the resulting FVIII:Cby FVIII:RP.

Zimmerman, et al., U.S. Pat. No. RE32011 disclose a human monoclonalantibody-based immunoaffinity two-step purification procedure. The firststep is adsorption of the FVIII:C/FVIII:RP complex from a human plasmasource, followed by a buffer wash, and subsequent desorption of FVIII:Cusing a calcium solution that elutes only FVIII:C (FVIII:RP remainsbound). The second step is concentration of the FVIII:C by adsorbing theeluate from step one to an aminohexyl-substituted agarose column with asubsequent calcium buffer wash. This results in a concentration of FVIIIof over 160,000-fold from plasma.

Several purification schemes utilize antibody affinity columns. Wood,W., et al., Nature, (1984), 312: 330-337 demonstrated anotherimmunoaffinity purification, using the C7F7 MAb which is specific to theFVIII:C 80 kD fragment. Rotblat, F., et al., Biochemistry, (1985), 24:4294-4300 also describe an immunoaffinity purification of FVIII:C ofover 300,000-fold from cryoprecipitate by polyelectrolyte purification,followed by affinity separation of a sepharose-anti-FVIIIR:Ag, and afinal adsorption to a FVIII:C specific MAb column. To date, the mostsuccessful purifications of Factor VIII from plasma or from recombinantsources have been accomplished by using murine monoclonal antibodiesspecific to either Factor VIII or von Willibrand Factor (see Zimmerman,et al, supra, U.S. Pat. No. RE32011).

Although monoclonal antibodies have been used successfully to obtain arelatively pure Factor VIII preparation, monoclonal antibodies can bepresent in the Factor VIII effluent because of leaching from the supportmatrix. This raises the possibility of antigenicity when the finalpreparation is introduced into animal systems, since murine monoclonalantibodies have been shown to be antigenic. A second disadvantage of theuse of monoclonal antibodies is the requirement of cell culturefacilities for producing the antibodies and the concomitant cost ofpurification and attachment onto a support matrix. And finally, thestability of the antibody binding site may not be amenable to therigorous conditions necessary to sanitize the column.

Affinity chromatography is one of the most efficient techniques forpurifying a protein from a complex mixture. With potential advantagesincluding high stability, efficiency, selectivity, and low price,peptides have been investigated as affinity ligands in thepharmaceutical industry. A recent approach for identifying such ligandsis to screen peptides from combinatorial peptide libraries (Baumbach, etal., BioPharm, (1992), 5: 23-35; Buettner, J., et al., Int. J. Pept.Prot. Res., (1996), 47: 70-83; Huang, P., et al., Biotechnol. & Bioeng.,(1995), 47: 288-297; Huang, P., et al., Bioorg. & Med. Chem., (1996), 4:699-708). It has been shown that by using the "divide-couple-recombine"approach (Furka, et al., Int. J. Pept. Prot. Res., (1992), 37: 487-493;Lam, et al., Nature, (1991), 354: 82-84; Houghten, et al., Nature,(1991), 354: 84-86), millions of unique peptides of a defined length maybe synthesized on resin beads. Each bead contains a unique peptidesequence. These library beads and their corresponding peptide sequencesare then exposed to a target protein. Among these millions of peptidesequences, the target protein may bind to several unique bead-sequences.Those beads and their corresponding sequences must be detected,isolated, and identified. Several detection systems, includingcolorimetric two-step methods (Buettner, et al., (1996), 47: 70-83;Houghten, et al. (1991); Lam, et al., J. Immunol. Meth., (1995), 180:219-223) as well as direct fluorescence detection methods (Meldal, etal., Proc. Nat. Acad. Sci., (1994), 91: 3314-3318; Meldal, et al., J.Chem. Soc. Perkin Trans., (1995) 1: 1591-1596; Needles, et al., Proc.Natl. Acad. Sci., (1993), 90: 10700-10704) have been used.

Another method of generating large libraries of peptides for affinityseparations is phage display. WO 95/18830, Inhibitors of human plasminderived from the kunitz domains, describes the selection of bindingdomains specific for human plasmin useful as inhibitors, includingapplications for drugs, for diagnostic reagents and for affinitypurification ligands. The claimed binding domains are chimeras of one ofthe three kunitz binding domains found in lipoprotein-associatedcoagulation inhibitor (LACI), a 39 kd protein. The binding domains areabout 58 amino acids with 7 to 11 amino acid substitutions generated bycombinatorial methods. No smaller peptides are used or claimed.

WO 97/35197, Purification of tissue plasminogen activator (tPA), claimsbinding domains useful for purification of the biological drug tPA,using the same technology as described in WO 95/18830 and WO 97/35196.In this application, the binding domains are 29 amino acids of which 8were combinatiorialized. One claimed sequence is from a peptide of 11amino acids, of which 8 residues were derived combinatorially from thebinding motifs of the other claimed domains, having the 11-mer sequence:Arg Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa. Specific residues areclaimed for each position (Xaa) for this presumed cyclic peptide.

It is apparent that other, more specific affinity peptides for bindingproteins of biological interest are needed.

SUMMARY OF THE INVENTION

We have discovered a group of small peptides characterized by theirability to bind and purify human Factor VIII. The invention is acomposition for binding Factor VIII comprising a peptide having anavailable Factor VIII binding domain having the sequence of SEQ ID NO:1, 2 or 3. These peptides bind Factor VIII while the peptide iscovalently linked onto a chromatographic support under the conditionsdescribed. The sequences of the more preferred peptides having a FactorVIII binding domain are Trp-His-Tyr-Tyr-His-Gly (WHYYHG),His-Ile-Gln-His-Tyr-His (HIQHYH), and His-Gln-Tyr-Gly-Tyr-His (HQYGYH);sequence identification numbers 1, 2 & 3, respectively. The peptideswere isolated and identified using a screening process described in U.S.Patent Application 08/438,331, incorporated herein by reference in itsentirety.

The invention also includes a method of purifying Factor VIII comprisingcontacting a solution containing Factor VIII with a Factor VIII-bindingsubstrate under conditions sufficient to bind Factor VIII to saidsubstrate, wherein said Factor VIII-binding substrate comprises one ormore peptides bound to said substrate, wherein said peptides areselected from the group consisting of SEQ ID NO:1, SEQ ID NO: 2, OR SEQID NO:3, and then eluting the bound Factor VIII.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a chromatogram showing the elution profile (absorbance at 280nm) of chromatographic separations (secondary binding assay) on thepeptide WHYYHG-A-TSK650M (SEQ ID NO: 1).

FIG. 2 is a chromatogram showing the elution profile (absorbance at 280nm) of chromatographic separations (secondary binding assay) on thepeptide HIQHYH-A-TSK650M (SEQ ID NO: 2).

FIG. 3 is a chromatogram showing the elution profile (absorbance at 280mn) of chromatographic separations (secondary binding assay) on thepeptide HQYGYH-A-TSK650M (SEQ ID NO: 3).

FIG. 4 is a chromatogram showing the overlaid elution profiles(absorbance at 280 nm) of chromatographic separations on the peptideWHYYHG-A-TSK650M (SEQ ID NO: 1). A) buffer blank injection with noprotein; B) & C) & D) are identical injections of KG2DEAE.

FIG. 5 is a chromatogram showing the overlaid elution profiles(absorbance at 280 nm) of chromatographic separations on the peptideHIQHYH-A-TSK650M (SEQ ID NO: 2). A) buffer blank injection with noprotein; B) & C) & D) are identical injections of KG2DEAE.

FIG. 6 is a chromatogram showing the overlaid elution profiles(absorbance at 280 nm) of chromatographic separations on the peptideHQYGYH-A-TSK650M (SEQ ID NO: 3). A) buffer blank injection with noprotein; B) & C) & D) are identical injections of KG2DEAE.

FIG. 7 shows SDS-PAGE and Western Blot analyses for the 2^(nd) and3^(rd) injection for the peptide affinity resin SEQ ID NO: 1 (WHYYHG).Lane designations: M=molecular weight markers; D=KG2DEAE hrFVIIIin-process fraction; K=KG2 hrFVIII highly purified; FT=flow-throughcolumn fraction; S=salt wash column fraction; E=elution column fraction.

FIG. 8 shows SDS-PAGE and Western Blot analyses for the 2^(nd) and3^(rd) injection for the peptide affinity resin SEQ ID NO: 2 (HIQHYH).Lane designations: M=molecular weight markers; D=KG2DEAE hrFVIIIin-process fraction; K=KG2 hrFVIII highly purified; FT=flow-throughcolumn fraction; S=salt wash column fraction; E=elution column fraction.

FIG. 9 shows SDS-PAGE and Western Blot analyses for the 2^(nd) and3^(rd) injection for the peptide affinity resin SEQ ID NO: 3 (HQYGYH).Lane designations: M=molecular weight markers; D=KG2DEAE hrFVIIIin-process fraction; K=KG2 hrFVIII highly purified; FT=flow-throughcolumn fraction; S=salt wash column fraction; E=elution column fraction.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Materials

Monoclonal antibodies directed to each hrFVIII chain, polyclonalantisera against the hrFVIII non-expressing BHK cell medium, and anin-process fraction of human Factor VIII (KG2DEAE) were obtained fromBayer Corporation, Biotechnology Division, Berkeley, Calif. Secondaryantibody conjugates and the dye substrates NBT/BCIP and Fast Red wereobtained from Pierce Chemical Company, Rockford, Ill. An in-processfraction of recombinant human Factor VIII (KG2DEAE) was obtained fromBayer Corporation, Berkeley, Calif. FMOC amino acids were fromNovabiochem, San Diego, Calif. All other chemicals were reagent grade orbetter.

HPLC

The secondary chromatography binding assays were performed upon aMichrom Bioresources HPLC (Auburn, Calif.). This binary system has theadded advantage of a second injection loop which was outfitted with a 5ml loop. This allowed addition of a third solvent during the HPLC run.Buffer A was 20 mM Imidazole, pH=7.0, 10 mM CaCl₂, 300 mM NaCl, 0.01%Tween 80. Buffer B is the same as buffer A but with 1M NaCl and 1MCaCl₂. Elution buffer (Buffer C) is the same as buffer A but with 40%ethylene glycol monomer and 250 mM CaCl₂. Absorbance was monitored at280 nm.

Library Primary Screen

An assay similar to the one used in U.S. patent application Ser. No.08/438,331 (incorporated herein by reference) was used to deduce thepeptides that bind human Factor VIII. Colorimetric methods are usuallybased upon a primary antibody and secondary antibody-conjugate system.Antibodies have the potential for being very specific and sensitive.However, antibody-based methods also have the potential forfalse-positive (yet specific) interactions due to the adsorption ofprimary antibodies and secondary antibody-conjugates to the peptidelibrary ligand(s) that do not bind the target protein. Therefore, atwo-step, subtractive method for identifying peptide ligands frompeptide libraries has been developed (U.S. patent application Ser. No.08/438,331; Buettner, J., 1996, supra). Briefly, the detecting antibodysystem is contacted with the on-resin peptide library and allowed toadsorb either specifically or non-specifically. The resin is washed toclear unbound detection reagents, then beads that have bound detectionreagents are identified by using a blue precipitating substrate. Thetarget protein is then allowed to contact the same library, withsubsequent contact with the same detection reagents. This time thetarget-specific beads are colored red with a precipitating substrate.Red beads are isolated manually and sequenced by classical techniques inthe literature (Lebl, et al., Biopolymers (Peptide Science), (1995), 37:177-198).

Secondary Chromatography Binding Assay

Confirmatory FVIII binding assays were performed in a columnchromatographic format on the HPLC described above. Batch synthesizedpeptide-resin (substitution at 100 μmoles/g) was loaded and washed asdescribed in Buettner, J., 1996, supra. The HPLC method injected thesample onto the column at 80 cm/hr linear velocity, allowed forflow-through of unbound protein, then stepped to 200 cm/hr for the washwith Buffer B (as described above), and then the bound protein elutedwith Buffer C (as described above). FIGS. 1, 2 and 3 show the resultsfrom contacting a known amount of human Factor VIII to each of thepreferred peptide-resins (sequence ID 1, 2 and 3, respectively).

    ______________________________________                                                 Load                                                                    rhFVIII Flow-Through Salt Wash Elute                                         Sequence ID Total μg Total μg Total μg Total μg                 ______________________________________                                        1) WHYYHG                                                                              10 μg 0.03        0.14    5.31                                      2) HIQHYH 10 μg 0.00 0.74 7.26                                             3) HQYGYH 10 μg 0.00 0.85 10.31                                          ______________________________________                                    

Recombinant FVIII was applied to each column as described above. Theendpoint of this secondary binding assay is to demonstrate binding ofthe target protein to the preferred peptide ligand; quantification ofbinding is by the presence or absence of Factor VIII immunologicalidentification (ELISA). ELISA quantitation utilized a polyclonalantisera as capture antibody adsorbed onto the plate well, applicationof each fraction allowed the Factor VIII to adhere to the captureantibody. The making and using of polyclonal and monoclonal antibodiesis well-know, and within the skill of one of ordinary skill in the art.See generally Antibodies, A Lab Manual, Harlow and Lane, eds., 1988,Cold Spring Harbor. Then a panel of 3 monoclonal antibodies were used toidentify the Factor VIII and detection of these antibodies with asecondary-antibody enzyme conjugate allowed accurate quantitation of theFactor VIII. The flow-through fractions from each injection show greaterthan 96% target protein depletion by these peptide ligands. The bindingavidity was found to be sufficiently strong enough to withstandchallenge from high salt buffer wash. The binding avidity was weakenough to permit recovery of target protein during the elution phase ofthe chromatography.

Demonstration of the binding of FVIII to the peptide resin can also bedemonstrated in a radioimmunoassay by radiolabeling the FVIII andmeasuring the adsorbed and desorbed radioactivity. For instance, ¹⁴ Clabeling by reductive methylation using sodium cyanoborohydride and ¹⁴C-formaldehyde is one such technique (see Jentoft et al., Methods inEnzymology 91: 570-579 (1983)). It will be apparent to one of ordinaryskill in the art that there are other techniques for assessing whetherthe FVIII has bound to the target peptide, such as fluorometric labelingand analysis, avidin/biotin, etc..

Modification of the six-mer peptides described herein may be made byconservative modifications and substitutions at positions within thepeptide (i.e., those that have a minimal effect on the binding of FVIIIto the peptide). Such conservative substitutions include those describedby Dayhoff in The Atlas of Protein Sequence and Structure 5 (1978), andby Argos in EMBO J., 8: 779-785 (1989). For example, amino acidsbelonging to one of the following groups represent conservative changes:

ala, pro, gly, gln, asn, ser, thr;

cys, ser, tyr, thr;

val, ile, leu, met, ala, phe;

lys, arg, his;

phe, tyr, trp, his; and

asp, glu.

EXAMPLE 1

Preparative hrFVIII Purification by Preferred Peptide Resins

Preparative batch synthesis of the preferred sequences was performedusing 10 g TSK resin (Toyopearl AF-Amino-650M, TosoHaas, Inc.,Montgomeryville, Pa.) as described in Buettner, J., 1996, supra. Foreach batch, a small amount of resin was loaded into 0.5 cm×5 cm HPLCcolumns to demonstrate affinity purification. Three injections of anin-process sample of human Factor VIII were contacted with each resinand fractions of flow-through, salt wash and elution were tested forbiological activity, protein profile by SDS-PAGE, and target proteinidentification by Western blot analyses, as described in Buettner, J.,1996, supra. Biological activity was defined as the ability of thecolumn fraction to reconstitute Factor VIII-depleted plasma to therebyactivate Factor X to Factor Xa, with quantitation of enzymatic activityby spectrophotometric measurement of cleaved substrate. (CoatestVIII:C/4, Chromogenix, Molndal, Sweden).

FIGS. 4, 5 and 6 show the purification chromatograms for binding of therhFVIII to SEQ IDs 1, 2 and 3, respectively. In each figure, the bottomHPLC trace is the buffer blank injection; the three other HPLC tracesare subsequent injections of the KG2DEAE sample. For each chromatogram,flow-through occurs from t=0-22 minutes; salt wash at t=26-34 minutes;elution at t=44-54 minutes. Each fraction (flow-through, salt wash andelution) was collected and assayed for Factor VIII activity by the abovedescribed Coatest assay.

    __________________________________________________________________________          Load  Flow-                                                                              Salt       Elute                                                KG2DEAE Through Wash Elute %                                                 Sequence Total IU Total IU Total IU Total IU Recovery Purification          __________________________________________________________________________    WHYYHG                                                                          inj 2 136IU 5IU 2IU 35IU 26% 1.15                                             inj 3 136IU 2IU 2IU 56IU 41% 1.75                                             inj 4 136IU 6IU 5IU 54IU 40% 1.64                                             HIQHVH                                                                        inj 2 136IU 1IU 4IU 42IU 31% 1.22                                             inj 3 136IU 1IU 7IU 52IU 38% 1.54                                             inj 4 136IU 1IU 9IU 45IU 33% 1.48                                             HQYGYH                                                                        inj 2 136IU 2IU 3IU 62IU 46% 1.00                                             inj 3 136IU 1IU 3IU 71IU 52% 1.53                                             inj 4 136IU 1IU 3IU 75IU 55% 1.47                                           __________________________________________________________________________

Each of the preferred peptide resins demonstrated quantitative captureof the human Factor VIII applied. As shown in the above data table, eachpeptide-resin released the biologically competent Factor VIII in theelution fraction under these specific conditions. For each injection ofthe sample, a purification of 1- to 2-fold was observed.

FIGS. 7, 8 and 9 show the SDS-PAGE and Western blot profiles for thesecond and third injection fractions from each of the preferred peptideresins. Each of the SDS-PAGE profiles from the flow-through, salt andelution are consistent within each column run, but are different fromthe other columns suggesting each resin has a different specificity orselectivity for the sample components. Western blot analyses indicatecomplete adsorption of the Factor VIII onto each preferredpeptide-resin, with preferential release in the elution fraction. BothSDS-PAGE and Western blot analyses confirm the biological assay datapresented above.

Other embodiments of the invention will become apparent to one ofordinary skill in the art. For instance, minor modifications of thesix-mer peptides disclosed herein that substitute, add or delete one ormore amino acid residues that are similar in biochemical behavior tothose specifically disclosed herein ("conservative substitutions") andthat result in similar FVIII binding will be apparent to one of ordinaryskill in the art. These conservative substitution variants come withinthe spirit and scope of the invention, as delimited by the claims below.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 3                                        - - <210> SEQ ID NO 1                                                        <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <221> NAME/KEY: PEPTIDE                                                       <222> LOCATION: (1)..(6)                                                      <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: Random             peptide library                                                          - - <400> SEQUENCE: 1                                                         - -       Trp His Tyr Tyr His Gly                                                    1          - #     5                                                   - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <221> NAME/KEY: PEPTIDE                                                       <222> LOCATION: (1)..(6)                                                      <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: Random              peptide library                                                          - - <400> SEQUENCE: 2                                                         - -       His Ile Gln His Tyr His                                                    1          - #     5                                                   - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <221> NAME/KEY: PEPTIDE                                                       <222> LOCATION: (1)..(6)                                                      <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: Random              peptide library                                                          - - <400> SEQUENCE: 3                                                         - -       His Gln Tyr Gly Tyr His                                                    1          - #     5                                                 __________________________________________________________________________

We claim:
 1. A composition for binding Factor VIII comprising a peptideconsisting of an available Factor VIII binding domain which is SEQ IDNO:
 1. 2. A composition for binding Factor VIII comprising a peptideconsisting of an available Factor VIII binding domain which is SEQ IDNO:
 2. 3. A composition for binding Factor VIII comprising a peptideconsisting of an available Factor VIII binding domain which is SEQ IDNO: 3.